Central to the lifecycle of any virus are the processes of genome replication and virion particle assembly. These two events must be exquisitely coordinated within the infected cell to maximize viral multiplication and avert cell-intrinsic defenses. Rotaviruses are non-enveloped, eleven-segmented, double-stranded RNA viruses that cause severe diarrheal disease in young children. These viruses are also attractive experimental models to study how viral genome replication and particle assembly are coordinated, because they perform these tasks in tandem during their lifecycles. Specifically, rotavirus assembly is thought to begin with the formation of pre-core replication intermediates (RIs) comprised of the viral RNA polymerase (VP1) and RNA capping enzyme (VP3) bound to plus-strand RNA replication templates. Following the addition of the shell protein (VP2), the capsid protein (VP6), and nonstructural proteins (NSP2 and NSP5), eleven pre-core RIs become a single, replicase-competent core RI within which plus-strand RNAs are converted into genome segments by VP1-mediated minus-strand RNA synthesis. This concerted replicase-assembly mechanism requires that the enzymatic activity of VP1 be tightly controlled by its interactions with core RI proteins. Yet, critical gaps in knowledge exist about the structural organization of pre-core RIs and core RIs, as these complexes have not yet been seen. Moreover, the multifaceted interactions among VP1 and its co-factors remain poorly defined. The overall objective of this application is to shed new light on the early stages of rotavirus assembly and genome replication through the detailed structural, functional, and genetic analysis of RIs and their protein constituents. In AIM 1, native pre-core RIs and core RIs will be isolated from rotavirus-infected cells and their never-before-seen macromolecular architectures will be deduced using immunoaffinity-capture cryo-electron microscopy and single-particle image reconstruction. In AIM 2, interaction interfaces among three core RI proteins (VP1, VP2, and NSP2) will be defined using in vitro minus-strand RNA synthesis, virus-like particle formation, and co-immunoprecipitation assays. Finally, in AIM 3, the effects of interactions involving NSP2 on core RI formation and function will be studied in the context of rotavirus-infected cells using trans-complementation and single-gene reverse genetic approaches, thereby illuminating the role of this nonstructural protein during the replicase-assembly process. This proposal is innovative because it investigates original ideas about rotavirus genome replication and virion assembly for which little information currently exists. The work is significant because it will foster the development of targeted, next-generation rotavirus vaccines and may enhance our ability to engineer viral vectors as therapeutic delivery vehicles to treat human diseases.